CA1225333A

CA1225333A - Antiviral agents

Info

Publication number: CA1225333A
Application number: CA000439449A
Authority: CA
Inventors: Tatsuo Ito; Kazunori Oba; Tomizo Niwa; Harumi Fukuyasu; Takashi Shomura; Yuzo Kazuno
Original assignee: Meiji Seika Kaisha Ltd
Current assignee: Meiji Seika Kaisha Ltd
Priority date: 1982-10-28
Filing date: 1983-10-21
Publication date: 1987-08-11
Also published as: US4536398A; EP0116690B1; JPS637529B2; DE3371867D1; JPS5978120A; EP0116690A1

Abstract

Abstract:

Antiviral agents Disclosed is an anti viral agent comprising as an active component a substance SF-2140 described in detail in the specification, and a pharmaceutically acceptable carrier.
Also disclosed is a method for treatment of viaral diseases comprising administering a pharmaceutically effective amount of the substance SF-2140.

Description

S3~33 Anti viral agents This invention relates to a novel anti viral agent. More particularly, it is concerned with a novel anti viral composition which comprises as an active ingredient an 5 antibiotic substance SF-2140.

This invention is described below in detail with reference to the accompanying drawings.

In the drawings;

Fig. 1 shows an ultra violet absorption spectrum of the 10 substance SF-2140 in methanol;
Fig. 2 shows an infrared absorption spectrum of the substance SF-2140 in KBr tablet; and Fig. 3 shows NOR spectrum of the substance SF-2140 measured with 100 MHz and denteriechloroform.

15 It has been previously found that an antibiotic substance named SF-2140 can be produced by cultivation of actinomycetes belonging to the genus Actinomadura in a nutrient medium as a result of studies on new and useful antibiotic substances having an antibacterial activity 20 against gram-positive and -negative bacteria, and also established physicochemicaland biological properties of Jo 533;~

the so-isolated substance SF-2140, as disclosed in Japanese Unexamined Patent Publication No. 85397/1982.

AS one example of the substance SF-2140-producing microorganisms belonging to the genus Actinomadura, there 5 is mentioned the strain SF-2140 isolated from a soil sample collected in Hyogo-prefecture, Japan in September, 1979.

This SF-2140 strain has the following morphological and Physiological properties:

10 I. Morphological properties Substrate Muslim extends well-branched and not disrupted under ordinary conditions. Aerial Muslim generally grows abundantly and arthrospore is well formed. Aerial Muslim forms simple branches which are 15 not clustered; no sporangium and sclerotium observed; a straight chain of spores observed all over the aerial Muslim; and no flagella spores observed. Electron microscopic examination shows spores are oval to cylindrical in shape and 0.4 to 0.6 x 0.6 to 1.3 mu in 20 size, long spore chains of scores or more are observed and spore surface is smooth.

2S;~33 II. Growth on various culture media Growth and Aerial Soluble-Culture medium color on reverse Muslim pigment Sucrose-nitrate Good, rusty Abundant, None ajar greenish blue white (a) (5 to) Glucose- Moderate, topaz Abundant, None asparagine ajar (3 no) pearl (3 cay Glycerol- Good, red Abundant, None asparagine ajar greenish blue white (a) (4 no) Starch ajar Good, dark red Abundant, None greenish blue white (a) (I pug) Oatmeal ajar Good, semi Abundant, None greenish blue white (a) (2 go) Yeast extract- Good, rusty Abundant, None malt extract brown (5 pug) white (a) ajar Tarzan ajar Good, deep Abundant, Wove brown (4 pi) pearl (3 be) Nutrient ajar Good, cocoa Poor, None brown (2 fob) white (a) The above results were obtained after cultivation at 28C
over 14 to 21 days. Color standards indicated in parentheses in the above Table were determined according to the color classification as taught in "Color Harmony Manual", Container's Corporation of America.

III. Physiological properties (1) Temperature range for growth (on starch ajar):
20 to 45C, good growth at 25 to 45C

(2) Liquefaction of gelatin: Positive AL I' 2~3~3

(3) Hydrolysis of starch Positive

(4) Peptonization of skim milk: Negative

(5) Coagulation of skim milk: Positive

(6) Melanin formation: Negative

(7) Nitrate reduction: Negative

(8) Salt resistance: Growth at 5 % or lower of Nikko, no growth at 7 % or higher.

IV. Utilization of carbon source Basal medium (0.5 % yeast extract, 0.1 % calcium carbonate and 1.5 ajar) containing 1 % each of the following carbon sources.

D-glucose D-fructose +
D-xylose +
L-arabinose +
D-mannitol +
L-rhamnose +
sucrose +
Instill ruffians +: utilized -: not utilized V. Whole cell analysis Methods taught by Becker et at (Apply. Microbial., 12, 421 423, 1964) and by MOP. Lechevalier et at (International 25 Journal of Systematic Bacteriology, 20, 435 - 443, 1970) were used for analysis.

~5333 (1) 2,6-Diaminopimolic acid in whole cells: mess type (2) Saccharides in whole cells: No Arabians and Zulus contained with a minor amount of Madras.

From the foregoing properties, the SF-2140 strain may morphologically resemble to that of the genus Streptornyces, but -the strain is to be clearly distinguished from the genus Streptomyces, in view of meso-2,6-diaminopimelic acid contained in whole cells, and also from the typical Nocardia genus, in view of no Arabians contained in whole cells. Based upon the system as taught by MOP. Lechevalier et at., swooper, the strain SF-2140 can be most reasonably regarded as belonging to the genus Actinomadura. Therefore, the strain SF-2140 has been named as Actinomadura spy SF-2140. The strain SF-2140 has been deposited under international deposit with accession number FORM 3P-386, dated September 9, 1980, in Fermentation Research Institute, Agency of Industrial Science & Technology, Ministry of International Trade & Industry, Japan.

As seen in other actinomycetes, the strain SF-2140 is susceptible to variation in nature and may be easily variable artificially for example, by using ultraviolet ray, X-ray, high frequency current, radiation, medicines and the like. Any variants and mutants of the strain SF-2140 may be usable for this invention if they have a productivity of the substance SF-2140. Moreover, any substance SF-2140-producing strains belonging to the genus Actinomadura may be employed for this invention.

For production of the antibiotic substance SF-2140, a SF-2140 substance-producing microorganism belonging to the genus Actinomadura is cultivated on a culture medium and -the substance SF-2140 is recovered from a cultured broth. More specifically, the said microorganism is cultivated in a culture medium containing those nutrients utilizable by ordinary microbes. As nutrient sources, there may be employed any well-known nutrients commonly used for cultivation of actinomycetes. For instance, 5 there may be employed as a carbon source, such as glucose, sucrose, starch, glycerol, corn syrup, molasses, soybean oil and the like. As a nitrogen source, there may be mentioned, for example, soybean meal, wheat embryo, meat extract, petunia, yeast extract, corn steep 10 liquor, ammonium sulfate, sodium nitrate and the like.
If necessary, there may be further incorporated an inorganic salt such as calcium carbonate, sodium chloride, potassium chloride, phosphates and so on and any organic or inorganic substance capable of promoting 15 growth of the strain and production of the substance SF-2140. Cultivation may generally be conducted according to conventional cultivation procedures for production of antibiotic substances and, particularly, submerged culture in a liquid medium is most preferable.
20 Cultivation may be conducted under aerobic condition, cultivation temperature is usually 25 to 37C, but favorably around 28C. Maximum production of the substance SF-2140 can be achieved in 2 to 6 days at both shaken culture and tank culture.

25 For assay of the substance SF-2140, bioassay ajar is used as a medium for assay and Vibrio percolance is as a test microbe. In this assay, the substance SF-2140 shows a linear relationship between logarithm of concentration and diameter of inhibition zone at 1000 mcg/ml to 125 30 mcg/ml and the diameter of inhibition zone is 22.0 to 14.6 mm, respectively, according to a paper disc plate method.

For recovery of the substance SF-2140 from a cultured broth, there may be applied for its extraction and 35 purification a synthetic adsorbent such as Amberlite 33.3 I (a Lra(l~nark avowal from~ohln&llass Co., USURY, Dunn IIP-~o (a trademark available from Mitsubishi Chemical Industries Lo Japan); a gel filtering agent such as Sephadex LH-20 (a trademark available from Pharmacia Fine Chemicals, Sweden);
S precipitation with hexane; extraction with a solvent, e.g. ethyl acetate : column chromatography with silica gel; and the like. More efficiently, Mazola and other solid materials may be filtered off from a cultured broth by using a filter aid, e.g. diatomaceous earth and then the active ingredient in the filtrate is adsorbed onto Dunn HP-20. The resin is washed with water and eluded with 50 % aqueous acetone. Active fractions of equates are concentrated under reduced pressure to remove the acetone. The concentrate is extracted with ethyl acetate, extract is concentrated to dryness under reduced pressure and the resultant residue is further purified by any optional combination of column chromatography with silica gel developed with chloroform-methanol, 30 Sephadex LH-20 and the like, thereby affording a highly purified substance SF-2140.

VI. Physico-chemical properties of the substance SF-2140 (1) Color and state: Colorless crystals, neutral substance (2) Melting print: 174 to 176C (with decomp.) (3) Elementary analysis: C: S9.54%, H: 5.63~, N: 7.59~
(4) Ultraviolet absorption spectrum (as shown in Fig. 1):
Maximum absorption at 222 no (Elm = 960), 258 no (so), 265 no (Elm = 228), 284 no (Elm = 174),294 no (Elm = 192).
No shift with acid or alkali observed.

, 3~33 (5) Infrared absorption spectrum in KBr:

As shown in Fig. 2 (6) Molecular weight (Mass spectrum): 360 (7) Molecular formula:

C18H20N2O6 as determined upon the elementary analysis and NOR spectrum.

(8) NOR spectrum (CDC3, 100 MHz, H-NMR spectrum):

As shown in Fig. 3.

(9) Specific rotation:

Do = +50.2 (Of methanol)

(10) Volubility:

Easily soluble: Lower alcohol, e.g. methanol and ethanol Soluble: Ethyl acetate, Bunsen, acetone, chloroform Substantially insoluble: Hexane, water

(11) Of value in silica gel thin layer chromatography:

Chloroform-methanol (5 : 1) 0.53 Ethyl acetate-benzene (2 : 1) 0.31 Acetone-benzene (2 : 1) 0.82

(12) Color reaction:

Lemiex-sulfuric acid reaction: Positive Ninhydrin reaction: Negative g

(13) Stability:

Stable under acidic to neutral condition, instable under alkaline condition.

The substance SF-2140 has been regarded as a novel substance. More specifically, the known substances "Trienin" journal of Antibiotics, 21, 611 - 615, 1968) and "Mycotrienin" (Journal of Antibiotics, 20, 32g - 333, 1967) have a relative resemblance to the substance SF-2140 with regard to the absorption pattern in ultraviolet absorption spectrum but they are yellow substances and quite different in elementary analysis and molecular weight from the SF-2140 substance. Thus, the substance SF-2140 can be definitely distinguished from the prior two substances. On the other hand, other known antibiotics, "Quinamycins A, s, C, D" (Journal of Antibiotics, 24, 353 - 359, 1971), which have respective molecular formulae, (C24H20N2olo~ Clown C24H20N216 and C~2H18N2Og, appear to have a relative resemblance to the SF-2140 substance having the above-defined molecular formula, but they can be definitely distinguished from the substance SF-2140 with regard to the color, ultraviolet, infrared and NOR spectra, specific rotation and others.

Also, the substance SF-2140 was found to show an antibacterial activity against various organisms as illustrated in the following Table 1.

~Z~33~3 Table l Minimum Inhibitory Test organisms Concentration (mcg/ml)*
Staphylococcus Ayers 209P 25 Staphylococcus Ayers Smith 100 Streptococcus focalize ATTICS 12.5 Bacillus anthrasis No. ll9 6.25 Escherichia golf No. 29 >100 Escherichia colt Wow RUN 823 50 Citrobacter frowned GUN 346>100 Salmonella tough 0-901-W 100 Salmonella enteritidis No. ll12.5 Sarcina lute 50 Shekel sonnet EYE type I>100 Klebsiella pneumonia PI 602>100 Proteus vulqaris OX-l9 25 Proteus Morgan Cowan >100 Pseudomonas aeruginosa MY 3829 >100 Pseudomonas epoch M-0527 100 * ajar dilution method As a planar structure of the substance, the substance SF-2140 has been determined to have the following chemical stutter:
OUCH
r owe COUCH
I

OH

~ZZ5~33 As a result of further studies on biological activities of the said substance SF-2140 made by the present inventors, it has been unexpectedly found that the substance SF-2140 can exert a potent anti viral activity, The present invention thus provides a new anti viral agent which comprises SF-2140 and a pear-mystical acceptable carrier.

The present invention also provides a method for treatment of viral diseases in humans by ad minis-toning the substance SF-2140.

The substances SF-2140 may be, illustratively speaking, produced, for example, as disclosed in the following Referential Example.

Referential Example Cultivation of SF-2140 strain:

As the seed strain, there was employed Actinomadura spy SF-2140 strain (FORM P-5704) and, as a seed vulture medium, there was employed a medium of 1.0 soluble starch, 1.0 % glucose, 0.5 % petunia, 0.2 meat extract, 0.3 % yeast extract, 0.2 % fine soybean meal and 0.2 %
calcium carbonate. Two or three platinum loops of the seed strain were inoculated to 20 ml of the seed culture medium in a 100 ml volume Erlenmeyer flask and cultivation was effected at 28C for 48 hours. The resulting seed culture was in each 4 ml portion inoculated to each 80 ml of the seed culture medium in l ) three 500 ml volume Erlenmeyer flasks and cultivation was effected at 28C for 4 hours.

The resulting seed culture was in each 4 ml portion inoculated to each 80 ml of a production medium in fifty 500 ml volume Erlenmeyer flasks. The production medium had composition of 1.5 glycerol, 1.0 glucose, 1.5 fine soybean meal, 0.1 % yeast extract, 0.1 % potassium hydrogen phosphate, 0.1 % magnesium sulfate, 0.2 % calcium carbonate and 0.0001 % cobalt chloride (pi 7.0 before sterilization). Cultivation was effected at 28C for 96 hours in a shaken culture manner. After completion of the cultivation, filtration was carried out by using as a filter aid diatomaceous earth to produce 3.2 lit. of the culture broth filtrate.

Purification of Substance S~-2140:

The culture broth (3.2 Lotte obtained as above was passed through a column of 300 ml of Dunn HP-20 (trade name, manufactured by Mitsubishi Chemical Industries Ltd.) to adsorb active ingredient. The column was washed with 1 lit. of water and then eluded with 50 aqueous acetone , thereby active ingredient being eluded in the end to Thea fractions, each being 200 ml fraction. The active fractions were combined, concentrated under reduced pressure to remove the acetone.

25 The concentrate (300 ml) was adjusted to pi 8.0 with lo Noah and extracted with 300 ml of ethyl acetate. The extract was concentrated under reduced pressure to dryness to give 443 my of an oily substance (a purity of about 8 I). The substance was dissolved in 3 ml of methanol, 50 ml of hexane were added to the resulting solution and the mixture was allowed to stand under ice-cooling over 1 hour to precipitate the active ingredient. after removal of the supernatant, the precipitate was dissolved in 3 ml of a mixture of chloroform and methanol (3 : 1), charged onto a column of 100 ml of silica gel C-200 (manufactured by Wake Junk Cage K. K.) and developed with a mixture of chloroform and methanol (30 : 1), thereby the active ingredient being eluded in the Thea to lath fractions, each being 15 ml fraction. These active fractions were combined and concentrated under reduced pressure to dryness to afford 152 my of the substance SF-2140 as a crude powder (a purity of about 35 %).

The crude powder (152 my) was dissolved in 0.5 ml of methanol, subjected to two sheets of thin layer chromatography with silica gel (manufactured by Merck &
Co., Inc., F254, 20 cm ax 20 cm), developed with a developing solvent of ethyl acetate and Bunsen (2 : 1) over 3 hours and then the substance SF-2140 portions therein were scraped out and extracted twice with each 50 ml of ethyl acetate. The extract was concentrated to dryness under reduced pressure to give 38 my of the 20 substance SF-2140 in a high purity of about 90 %.

In 2 ml of methanol were dissolved 30 my of the substance SF-2140, the resulting solution was charged onto a column K o--`` of 150 ml of Sephadex LH-20 us wow Pharmacia Fine Chemicals Co.) previously packed with methanol and then developed with methanol. Active fractions were obtained as the Thea to sty fractions in each 5 ml fraction. These active fractions were combined and concentrated to dryness under reduced pressure to give 12 my of the substance SF-2140 as colorless crystals.

Biological tests are shown below for illustrating anti viral effects of the present anti viral agent:

~S3~3

- 14 -Test 1 The substance SF-2140 was tested for anti viral activity against influenza virus.

(1) Test virus strains:

(a) Influenza virus Appear (b) Al/FM-l ( c ) A2/Adachi (d) " " B/Lee (e) " " Horse/Miami (2) Test method:

Proliferation inhibiting activity against virus and virucidal activity were measured according to a chorioallantoic membrane culture method. Namely, chorioallantoic membrane was isolated from 15-day-old embryonated egg and a given membrane piece thereof (30 mm x 30 mm) was placed into a culture test tube containing Hanks' solution, said solution containing a prescribed concentration of the substance SF-2140. Each of the above-indicated influenza virus strains was inoculated to said solution and shaken culture was conducted at 36C for 48 hours. Thereafter, each culture was measured for RBC of Asian agglutination ability and 50 % proliferation inhibiting concentration against virus was calculated therefrom. Also, virus proliferation inhibition index was determined by dividing 50 % toxicity concentration of the substance SF-2140 against chorioallantoic membrane by 50 proliferation inhibiting concentration. On the other hand, each virus strain was contacted and admixed with Hanks' solution containing a prescribed concentration of the substance SF-2140 and incubation was conducted at 25C for 2 hours. Then, the incubated broth was diluted to a proper concentration and ~Z~333

- 15 -virus proliferation inhibiting concentration and proliferation inhibition index were calculated in the same manner as mentioned above to assign them as 50 %
virucidal concentration and virucidal index, respectively.

Further, 50 % membrane-disintegration (cytotoxic) concentration was measured under the same conditions as defined above. Namely, 4 membrane pieces were incubated at an optional test compound concentration for 24 hours and dyed with 5 % Try pan Blue to determine life or death of membrane cells.

to) Test results:

The results are summarized in the following Table 2.

Table 2 Concentration (mc~/ml) Virus 50 % 50 % Virus 50 Treatment strain Toxicity proliferation Virucidal gone. inhibiting gone.
gone.
A PRY >1000 6.303.2 (>158.7)* (>312.5)**
Al/FM-l " 50.5 4.6 (> 20.0)* (>217.4)**
SF-2140 A /Adachi " 100 58.8 2 (> 10.0)* (> 17.0)**
B/Lee " >20046.4 (> 21.6)**
Horse/Miami " 17.7 4.6 (> 56.5)* (>2I7.4)**
Inhibition index 50% inhibitor conch.

Virucidal Index 50% inhibitory conch.

r~d5i 33 3

- 16 -As apparent from the above results, the substance SF-2140 showed a proliferation inhibiting activity against the influenza virus strains, Appear, Al/FM-l, A2Adachi and Horse/Miami.

Also, the substance SF-2140 was seen to exhibit a potent virucidal activity against all test virus in virucidal activity determination; 200 or higher against the Appear, Al/FM-l and Horse/Miami strains and 15 or higher against even the A2/Adachi and B/Lee strains.

Test 2 Anti viral activities of the substance SF-2140 and Amantadine (a comparative example) in mice infected with influenza virus Appear strain were tested. The substance SF-2140 and Amandatine were orally administered to mice in = 10) immediately after infection of the virus and thereafter once a day for five days with the dose as prescribed in Table 3. Results are shown together in Table 3.

Table 3 Treatment Dose MUD Prolong rate Survival SF-2140 125mg/kg >13.1 >103 60 crystal " 62.5mg/kg _13.5 >106 70 Amantadine 250mg/kg>13.6 >107 60 Hal lo CMC 0.15ml >12.7 lo 30 Miss Virus: LO
lkg9cm2/lOmin. (Inhalation) MUD mean survival days I

Test 3 Anti viral activities of the substance SF-2140 and Amantadine in mice infected with influenza virus Appear strain were tested. The substance SF-2140 and Amantadine 5 were intraperitoneally administered immediately after infection of the virus and thereafter once a day for five days with the dose as prescribed in Table 4. Results are shown together in Table 4.

Table 4 Disagreed of Lesion Inhibition Treatment(mg/kg) percent of LO consolidation (~) crystal 36.356.7 " 5014.5/40 36.356.7 " 2517.0/40 42.549~2 Amantadine Hal 10015.5/40 38.853.8 " 5019.5/40 48.841.8 Virus control - 33.5/4083.8 Test 4 10 Serum level of the substance SF-2140 after intro-peritoneal administration in mice (n = 5) was observed to obtain the results shown in Table 5.

~2~5;333 Table 5 Time after Concentration (mcg/ml):
administration (min~Biological assay Dose- 200mg/kg (1% CMC) Test 5 Serum level of the substance SF-2140 after oral administration in mice (n = 3) was observed to obtain the results shown in Table 6.

Table 6 Time after Concentration (mcg/ml):
administration (mix) HPLC

Dose: 125mg/kg I Arabic gum) cute toxicity Acute toxicity (LD50 value) of the substance SF~2140, the active ingredient in the present anti viral agent was not less than 500 mg/kg in mice via intraperitoneal ~S3~3 administration. This LD50 value demonstrates that the substance SE-2140 can be safely applied as an antivlral agent to human beings and other animals.

Administration and dose:

5 The present anti viral agent may be formulated in various pharmaceutical composition forms according -to conventional preparation techniques. The pharmaceutical composition may be of any preparation forms for oral and parenteral administration including aerosol treatment;
typically, inhalant, capsules, tablets, syrups, emulsions, aqueous suspensions, solutions or suspension for injection and so on, but it is preferable to orally administer the present agent formulated in an inhalant.

The usual dosage of this anti viral agent may be varied depending upon the severity of diseases, the weight and age of the patients being treated and other factors, but the active substance may be usually administered in the range of 0.05 to 25 g per day and in two to four divided doses per day.

Formulation Example 5 g of the substance SF-2140 was dissolved into Dixon and 15 g of PUP (polyvinylpyrrolidone, molecular weight:
about 10,000) into water. These were respectively freeze-dried to obtain powdery products, which were mixed together to obtain a mixed powdery product. The powdery product was then diluted with water to have a concentration of 0.3 I, thereby preparing an inhalant for a aerosol treatment.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An antiviral agent which comprises a substance SF-2140 represented by the following formula:

as an active component, and a pharmaceutically acceptable carrier.

2. An agent as claimed in claim 1 in the form of an inhalant.

3. An agent as claimed in claim 1 or 2 in dosage unit form of 2 to 4 doses containing 0.05 to 25 g.